The long-term goal of this proposal is to understand the biological role of the hemochromatosis disease gene, termed HFE, which carries one of the most common mutations currently known. The objectives are to unravel the product's functional niche in iron circuitry and to understand the molecular mechanisms through which it maintains iron balance in enterocytes and cells of the reticuloendothelial system. HFE is a unique member of the highly divergent class Ib gene family and as such, its expression in the mouse appears to be relegated to macrophages and enterocytes where it presumably exerts its biological effect(s). Unusual characteristics arising from our preliminary investigations include HFE's cell-surface expression even in the absence of beta2m and its partial dependence on TAP transport. In order to understand HFE's biological role, we have focused on three specific aims related to HFE's structure, expression (both in vitro and in vivo) and its relationship to intestinal iron flux and TNF-alpha responsiveness. Specifically, we will determine the elements required for HFE cell-surface expression (beta2m and TAP) and will determine the nature of bound self-peptide which, if specific, could herald a novel mechanism underlying HFE folding and transport. With respect to expression, we will analyze the kinetics of HFE's intracellular processing, paying particular attention to both its partial interaction with additional protein molecules (receptors or transporters) and to its possible subcellular routing and localization. Emanating from our preliminary data demonstrating increased HFE staining an a concomitant increase in intraepithelial lymphocytes staining positive for TNF-alpha in iron-loaded mice, we will continue to focus on the small intestine following iron challenge and will attempt to determine TNF-alpha's role in the maintenance of normal ferrokinetics within the context of control and knockout mouse model systems. Our hypothesis is that HFE, as part of a larger complex, undergoes up-regulation in the intestine in response to iron leading to an increase in macrophages and intestinal TNF-alpha which is directly related to enterocyte differentiation and iron flux.